The success of solid organ transplantation is limited by the toxicity of chronic immunosuppressive drugs and late graft loss due to chronic rejection. These problems would be overcome by induction of donor-specific tolerance. Establishment of mixed allogeneic chimerism using non-myeloablative conditioning achieves this goal in mice with minimal toxicity. Achievement of mixed chimerism requires a method of overcoming the pre- existing T cell barrier to donor marrow engraftment. This can be achieved by combining bone marrow transplantation (BMT) with costimulation blockade using anti-CD154 mAb. However, clinical use of anti-CD154 is associated with thromboembolic complications, and efficacy in large animals is limited. Thus, alternative approaches are needed. The goal of the studies proposed herein is to establish a system of in vivo delivery of small interfering (si)RNAs specifically into activated T cells as a novel therapeutic approach to inducing tolerance to allogeneic bone marrow grafts, thereby achieving durable mixed chimerism and subsequent central deletional tolerance. We hypothesize that in vivo delivery of siRNAs silencing transcripts that are critical for T cell activation, proliferation, and/or survival into T cells at the time of allogeneic BMT will result in anergy and deletion specifically of pre-existing donor-reactive T cells. Mixed allogeneic chimerism and central deletion of newly developing T cells will assure life-long donor-specific tolerance. Dr. Lieberman and colleagues have developed a method to introduce siRNAs specifically into activated lymphocytes using a fusion protein composed of domains 1 and 2 of murine ICAM-1 linked to an siRNA-binding protamine peptide. Since ICAM-1 encounters a high affinity form of its ligand LFA-1 only on activated lymphocytes, this construct will be used to target siRNAs to recipient T cells recognizing donor alloantigens in association with BMT. We aim to: 1) Demonstrate specific binding of the ICAM-1-protamine fusion protein and determine efficiency of delivery and knockdown in cultured cells;2) Validate and characterize the specificity and efficiency of binding, as well as the level of knockdown induced by the ICAM-1 construct-siRNA complex in vivo;and 3) Test the hypothesis that specific silencing of RasGRP1, cyclin D1, and bcl-xL in activated T cells is sufficient to tolerize donor-reactive T cells, permitting achievement of durable mixed chimerism and subsequent central tolerance. These studies will lead us toward a novel, clinically applicable strategy to achieve durable mixed chimerism and tolerance. PUBLIC HEALTH RELEVANCE: The proposed studies will lead us toward a novel, clinically applicable strategy to achieve durable mixed chimerism and organ allograft tolerance, avoiding the need for chronic immunosuppressive therapy and its attendant toxicities.